The photoreceptor PDE6 gamma subunit (Pg) is a critical regulatory molecule in rod and core photoreceptors cells and is also an important player in normal retinal development. The focus of the proposed research is to understand the molecular interactions of Pg, which regulates G protein-coupled receptor (GPCR) signaling in the visual system. This work will involve two Specific Aims: (1) To identify the interaction interfaces of the full-length PDEg molecule and the transducin alpha subunit (Gat) in the "GTP signaling state," in the "GDP-AIF4-transition state," and in the "RGS9/Gb5 transition state" complex. These experiments will be performed utilizing two complimentary approaches: (a) A systematic photolabel transfer to native and recombinant Gat from full-length PDEg photoprobes will be utilized to define the PDEg interaction sites on Gat complexed with GatGTPgS, GatGDP-AIF4- and in the full-length GatGDP-AIF4- /RGS9/Gb5 transition state complex using purified proteins followed by proteolytic cleavage and mass spectrometry analysis;(b) The solution structures of PDEg complexed with GatGTPgS, GatGDP-AIF4-, and recombinant Gat (Chi-8HN) will be determined by NMR spectroscopy utilizing 2H/13C/15N labeled proteins. (2) To identify the interaction surfaces between the PDE6 gamma subunit (PDEg), cyclic GMP and the PDE6 catalytic heterodimer subunits (PDEg). These experiments will be performed utilizing the following approaches: (a) The interaction site(s) on PDEg GAF domains that interact with the PDEg polycationic region will be identified through systematic photolabel transfer from PDEg photoprobes to PDEg, followed by proteolytic cleavage and mass spectrometry analysis;(b) The nature and modulation by PDEg of the cyclic GMP (cGMP) binding to the allosteric sites on the PDEg heterodimer will be elucidated utilizing non-radiolabeled and radiolabeled cGMP photolabeling experiments followed by proteolytic cleavage, SDS-PAGE and mass spectrometry analysis;(c) The NMR solution structure of the full-length PDEg complexed with the catalytic core of a PDE5/6 chimera will be determined using 2H/13C/15N labeled proteins. This work will provide important new knowledge in understanding regulation of GPCR-mediated signal transduction mechanisms in the visual system and the molecular interactions that are abrogated, leading to various forms of stationary night blindness in humans.